Expression and regulation of the human beta-defensins hBD-1 and hBD-2 in intestinal epithelium

The intestinal epithelium forms a physical barrier to limit access of enteric microbes to the host and contributes to innate host defense by producing effector molecules against luminal microbes. To further define the role of the intestinal epithelium in antimicrobial host defense, we analyzed the expression, regulation, and production of two antimicrobial peptides, human defensins hBD-1 and hBD-2, by human intestinal epithelial cells in vitro and in vivo. The human colon epithelial cell lines HT-29 and Caco-2 constitutively express hBD-1 mRNA and protein but not hBD-2. However, hBD-2 expression is rapidly induced by IL-1alpha stimulation or infection of those cells with enteroinvasive bacteria. Moreover, hBD-2 functions as a NF-kappaB target gene in the intestinal epithelium as blocking NF-kappaB activation inhibits the up-regulated expression of hBD-2 in response to IL-1alpha stimulation or bacterial infection. Caco-2 cells produce two hBD-1 isoforms and a hBD-2 peptide larger in size than previously described hBD-2 isoforms. Paralleling the in vitro findings, human fetal intestinal xenografts constitutively express hBD-1, but not hBD-2, and hBD-2 expression, but not hBD-1, is up-regulated in xenografts infected intraluminally with Salmonella. hBD-1 is expressed by the epithelium of normal human colon and small intestine, with a similar pattern of expression in inflamed colon. In contrast, there is little hBD-2 expression by the epithelium of normal colon, but abundant hBD-2 expression by the epithelium of inflamed colon. hBD-1 and hBD-2 may be integral components of epithelial innate immunity in the intestine, with each occupying a distinct functional niche in intestinal mucosal defense.     

Giardia lamblia: regulation of secretory vesicle formation and loss of ability to reattach during encystation in vitro

Encystation of Giardia lamblia is required for survival outside the host, as well as for initiation of new infections. Previously, we induced cultured G. lamblia trophozoites to encyst in vitro for the first time. During encystation, we observed the appearance of a new class of large secretory vesicle (encystation-specific vesicle; ESV) within which cyst antigens are concentrated and transported to the nascent wall. The present kinetic and physiologic studies now show that ESV are the earliest morphologic change observed in encystation. Expression of ESV, as well as subsequent encystation, are regulated by exposure to bile at the slightly alkaline pH which is typical of the human intestinal tract. ESV formation appears to be less stringently regulated than formation of water-resistant cysts because omission of either encystation stimuli or alkaline pH preferentially inhibits encystation. Since cysts do not attach, we asked when in encystation this physiologic transition occurs. We found that most encysting trophozoites remain attached until they begin to round up (greater than 24 hr). However, if they are made to detach, as early as 12 hr in encystation, well before they round up, they are defective in the ability to reattach. If trophozoites also become less able to reattach to the intestinal epithelium early in encystation in vivo, this would increase their exposure to lumenal encystation stimuli and promote encystation. These studies have provided new insights into the complex sequence of morphologic and physiologic alterations which occur during encystation of G. lamblia in vitro and their regulation by host intestinal factors.     

Polymeric immunoglobulin receptor in intestinal immune defense against the lumen-dwelling protozoan parasite Giardia

The polymeric Ig receptor (pIgR) is conserved in mammals and has an avian homologue, suggesting evolutionarily important functions in vertebrates. It transports multimeric IgA and IgM across polarized epithelia and is highly expressed in the intestine, yet little direct evidence exists for its importance in defense against common enteric pathogens. In this study, we demonstrate that pIgR can play a critical role in intestinal defense against the lumen-dwelling protozoan parasite Giardia, a leading cause of diarrheal disease. The receptor was essential for the eradication of Giardia when high luminal IgA levels were required. Clearance of Giardia muris, in which IgA plays a dominant role, was severely compromised in pIgR-deficient mice despite significant fecal IgA output at 10% of normal levels. In contrast, eradication of the human strain Giardia lamblia GS/M, for which adaptive immunity is less IgA dependent in mice, was unaffected by pIgR deficiency, indicating that pIgR had no physiologic role when lower luminal IgA levels were sufficient for parasite elimination. Immune IgA was greatly increased in the serum of pIgR-deficient mice, conferred passive protection against Giardia, and recognized several conserved giardial Ags, including ornithine carbamoyltransferase, arginine deiminase, alpha-enolase, and alpha- and beta-giardins, that are also detected in human giardiasis. Corroborative observations were made in mice lacking the J chain, which is required for pIgR-dependent transepithelial IgA transport. These results, together with prior data on pIgR-mediated immune neutralization of luminal cholera toxin, suggest that pIgR is essential in intestinal defense against pathogenic microbes with high-level and persistent luminal presence.     

The protective potency of probiotic bacteria and their microbial products against enteric infections-review

The intestinal environment accommodates a wide range of contents ranging from harmless beneficial dietary and microbial flora to harmful pathogenic bacteria. This has resulted in the development of highly adapted epithelial cells lining the intestine. This adaptation involves the potential of crypt cells to proliferate and to constantly replace villous cells that are lost due to maturity or death. As a result, the normal intestinal epithelial integrity and functions are maintained. This phenomenon is eminent in intestinal defense whereby the intestinal epithelial cells serve as a physical barrier against luminal agents. The protection against agents in the gut lumen can only be effective if the epithelium is intact. Restitution of the damaged epithelium is therefore crucial in this type of defense.     

Recent insights into the mucosal reactions associated with Giardia lamblia infections

Giardia lamblia is an intestinal protozoan parasite infecting humans and various other mammalian hosts. The most important clinical signs of giardiasis are diarrhoea and malabsorption. Giardia lamblia is able to undergo continuous antigenic variation of its major surface antigen, named VSP (variant surface protein). While intestinal antibodies, and more specifically anti-VSP IgA antibodies, were proven to be involved in modulating antigenic variation of the parasite the participation of the local antibody response in control of the parasite infection is still controversial. Conversely, previous studies based on experimental infections in mice showed that cellular immune mechanisms are essential for elimination of the parasite from its intestinal habitat. Furthermore, recent data indicated that inflammatory mast cells have a potential to directly, or indirectly, interfere in duodenal growth of G. lamblia trophozoites. However, this finding was challenged by other reports, which did not find a correlation between intestinal inflammation and resistance to infection. Since intestinal infiltration of inflammatory cells and/or CD8+T-cells were demonstrated to coincide with villus-shortening and crypt hyperplasia immunological reactions were considered to be a potential factor of pathogenesis in giardiasis. The contribution of physiological factors to pathogenesis was essentially assessed in vitro by co-cultivation of G. lamblia trophozoites with epithelial cell lines. By using this in vitro model, molecular (through surface lectins) and mechanical (through ventral disk) adhesion of trophozoites to the epithelium was shown to be crucial for increased epithelial permeability. This phenomenon as well as other Giardia-induced intestinal abnormalities such as loss of intestinal brush border surface area, villus flattening, inhibition of disaccharidase activities, and eventually also overgrowth of the enteric bacterial flora seem to be involved in the pathophysiology of giardiasis. However, it remains to be elucidated whether at least part of these pathological effects are causatively linked to the clinical manifestation of the disease.     

Uses and limitations of monoclonal antibodies to Giardia lamblia-specific 66-kDa copro-antigen in copro-immunodiagnosis of giardiasis

Abstract Antigen-capture enzyme-linked immunosorbent assay for the detection of Giardia lamblia -specific antigen in stool eluates from clinical subjects employing monoclonal antibody directed at 66-kDa G. lamblia copro-antigen has been evaluated. The G. lamblia copro-antigen was detected in 67% (31 of the 46 cases) of stool eluates from clinical cases, while none of the stool eluates from subjects with other intestinal parasites or from apparently healthy individuals, had detectable levels of G. lamblia copro-antigen. Monoclonal antibodies secreted by clones B4C5 and D3F4 recognised the periodate-sensitive and -insensitive epitopes of 66-kDa G. lamblia specific copro-antigen, respectively. Eight (73%) of the 11 symptomatic cases of giardiasis had trypsin-/periodate-sensitive epitopes of 66-kDa copro-antigen while 9 (92%) of 11 of the symptomatic cases and asymptomatic G. lamblia cyst carriers had trypsin-sensitive periodate-insensitive G. lamblia specific copro-antigen. The data tend to suggest that detection of periodate-insensitive epitopes of G. lamblia copro-antigen would indicate the presence of the parasite while the detection of periodate sensitive epitopes of G. lamblia copro-antigen would suggest symptomatic active giardial infection.     

Autophagy in the intestinal epithelium regulates Citrobacter rodentium infection

Autophagy, a ubiquitous degradation pathway, is important for the survival and homeostasis of cells. Previous studies have demonstrated the role of autophagy in host defense against bacterial infection, but the importance of autophagy in the intestinal epithelium for the regulation of bacterial infection has not been fully elucidated. In this study, we showed that the essential autophagy protein Atg7 is required for resistance to Citrobacter rodentium infection in the intestinal epithelium. Infected mice in which Atg7 had been conditionally deleted from the intestinal epithelium exhibited greater clinical evidence of disease and higher expression levels of pro-inflammatory cytokine mRNA in the large intestine. Moreover, C. rodentium clearance was reduced in the Atg7 conditional knockout mice. These results demonstrate that autophagy in intestinal epithelial cells plays an important role in host defense against C. rodentium infection and the regulation of C. rodentium infectious colitis.     

Nerves and secretory mechanisms

Summary The intestinal epithelium is a barrier which impedes penetration of the host's internal environment. One host defense mechanism is chloride secretion. Intrinsic neural reflexes within the submucous plexus regulate chloride secretion, a key process in lubrication and in flushing out the luminal contents. Axon reflexes through extrinsic primary afferents break the epithelial barrier, allowing foreign antigens and microbes to stimulate an immune response that can modulate activity of intrinsic neural reflexes and amplify epithelial chloride secretion.     

Giardia lamblia: electrophysiology and ultrastructure of cytopathology in cultured epithelial cells

The pathogenicity of Giardia lamblia is a subject of debate. Some studies of human biopsy material have mentioned the presence of trophozoites inside the intestinal mucosa, while in others, flagellates have only been found attached to the epithelium. To study the possible cytopathic effects of G. lamblia cultured under axenic conditions, trophozoites of the human 1/Portland and WB strains were placed in contact with monolayers of Madin Derby Canine Kidney cells, a well characterized cell strain with morphological and functional properties similar to those of a transporting epithelium. After 24 and 48 hr of interaction, the effect of the parasite on epithelial cells was assessed by transmission, scanning, and freeze fracture electron microscopy. In addition, the possible action of living trophozoites and sonicates of G. lamblia on the transepithelial resistance of MDCK monolayers mounted in Ussing chambers was analyzed for periods varying up to 48 hr. The results demonstrate that G. lamblia trophozoites do not invade epithelial monolayers. Furthermore, the parasites fail to produce cytoplasmic changes on target cells and have no effect on transepithelial resistance as judged both electrophysiologically and by the failure to open the occluding junctions that bind together epithelial cells. Damage induced by the parasites to cultured cells was limited to focal distortion or depletion of microvilli at the site of adhesion, which may progress to leave circular areas devoid of microvilli, different from the adhesion marks reported by others for G. muris. Therefore, under the in vitro conditions described here, giardias showed no toxic or invasive effect.     

Molecular interactions between bacteria, the epithelium, and the mucosal immune system in the intestinal tract: implications for chronic inflammation

In the last few years, advances in immunology, metabolomics and microbial ecology have shown that the contribution of the intestinal microbiota to the overall health status of the host has been so far underestimated. In this context, intestinal epithelial cells play a crucial role in the maintenance of intestinal homoeostasis. Indeed, at the interface between the luminal content and host tissues, the intestinal epithelium must integrate pro- and anti-inflammatory signals to regulate innate and adaptative immune responses, i.e. to control inflammation. However, under the influence of environmental factors, disturbance of the dialog between enteric bacteria and epithelial cells contributes to the development of chronic inflammation in genetically susceptible hosts. The present review covers the state of knowledge of the host response, especially in intestinal epithelial cells, to enteric bacteria, including colitogenic and probiotic bacteria. It also seeks to give an overview of potential regulatory mechanisms involved in the maintenance of intestinal homeostasis, and discusses the clinical implications for inflammatory bowel diseases.     

A bacterial effector targets Mad2L2, an APC inhibitor, to modulate host cell cycling

The gut epithelium self-renews every several days, providing an important innate defense system that limits bacterial colonization. Nevertheless, many bacterial pathogens, including Shigella, efficiently colonize the intestinal epithelium. Here, we show that the Shigella effector IpaB, when delivered into epithelial cells, causes cell-cycle arrest by targeting Mad2L2, an anaphase-promoting complex/cyclosome (APC) inhibitor. Cyclin B1 ubiquitination assays revealed that APC undergoes unscheduled activation due to IpaB interaction with the APC inhibitor Mad2L2. Synchronized HeLa cells infected with Shigella failed to accumulate Cyclin B1, Cdc20, and Plk1, causing cell-cycle arrest at the G2/M phase in an IpaB/Mad2L2-dependent manner. IpaB/Mad2L2-dependent cell-cycle arrest by Shigella infection was also demonstrated in rabbit intestinal crypt progenitors, and the IpaB-mediated arrest contributed to efficient colonization of the host cells. These results strongly indicate that Shigella employ special tactics to influence epithelial renewal in order to promote bacterial colonization of intestinal epithelium.     

Giardia immunity--an update

Giardia lamblia is a flagellated protozoan that causes watery diarrhea worldwide but the mechanisms of pathogenicity and the major host defenses against Giardia infection are not well characterized. The recent sequencing of the G. lamblia genome and the development of methods for genome-wide analyses of gene expression have made it possible to characterize the host-parasite interaction more fully. It is becoming clear that the host defense against a Giardia infection involves several different immunological and non-immunological mucosal processes.     

Arginine Consumption by the Intestinal Parasite Giardia intestinalis Reduces Proliferation of Intestinal Epithelial Cells

In the field of infectious diseases the multifaceted amino acid arginine has reached special attention as substrate for the host´s production of the antimicrobial agent nitric oxide (NO). A variety of infectious organisms interfere with this part of the host immune response by reducing the availability of arginine. This prompted us to further investigate additional roles of arginine during pathogen infections. As a model we used the intestinal parasite Giardia intestinalis that actively consumes arginine as main energy source and secretes an arginine-consuming enzyme, arginine deiminase (ADI). Reduced intestinal epithelial cell (IEC) proliferation is a common theme during bacterial and viral intestinal infections, but it has never been connected to arginine-consumption. Our specific question was thereby, whether the arginine-consumption by Giardia leads to reduced IEC proliferation, in addition to NO reduction. In vitro cultivation of human IEC lines in arginine-free or arginine/citrulline-complemented medium, as well as in interaction with different G. intestinalis isolates, were used to study effects on host cell replication by MTT assay. IEC proliferation was further analyzed by DNA content analysis, polyamine measurements and expressional analysis of cell cycle regulatory genes. IEC proliferation was reduced upon arginine-withdrawal and also in an arginine-dependent manner upon interaction with G. intestinalis or addition of Giardia ADI. We show that arginine-withdrawal by intestinal pathogens leads to a halt in the cell cycle in IECs through reduced polyamine levels and upregulated cell cycle inhibitory genes. This is of importance with regards to intestinal tissue homeostasis that is affected through reduced cell proliferation. Thus, the slower epithelial cell turnover helps the pathogen to maintain a more stable niche for colonization. This study also shows why supplementation therapy of diarrhea patients with arginine/citrulline is helpful and that citrulline especially should gain further attention in future treatment strategies.     

The host selects mucosal and luminal associations of coevolved gut microorganisms: a novel concept

Along the human gastrointestinal tract, microorganisms are confronted with multiple barriers. Besides selective physical conditions, the epithelium is regularly replaced and covered with a protective mucus layer trapping immune molecules. Recent insights into host defense strategies show that the host selects the intestinal microbiota, particularly the mucosa-associated microbial community. In this context, humans coevolved with thousands of intestinal microbial species that have adapted to provide host benefits, while avoiding pathogenic behavior that might destabilize their host interaction. While mucosal microorganisms would be crucial for immunological priming, luminal microorganisms would be important for nutrient digestion. Further, we propose that the intestinal microorganisms also coevolved with each other, leading to coherently organized, resilient microbial associations. During disturbances, functionally redundant members become more abundant and are crucial for preserving community functionality. The outside of the mucus layer, where host defense molecules are more diluted, could serve as an environment where microorganisms are protected from disturbances in the lumen and from where they can recolonize the lumen after perturbations. This might explain the remarkable temporal stability of microbial communities. Finally, commensals that become renegade or a decreased exposure to essential coevolved microorganisms may cause particular health problems such as inflammatory bowel diseases, obesity or allergies.     

Diversity of tight junctions (TJs) between gastrointestinal epithelial cells and their function in maintaining the mucosal barrier

The gastrointestinal epithelium, which is covered by a single layer of epithelial cells, including enterocytes, intraepithelial lymphocytes, goblet cells, microfold cells, and dendritic cells, serves as a protective barrier separating luminal contents from the underlying tissue compartments. The epithelium plays an important role in the first line of host defense against a variety of pathogens, as well as maintaining the homeostasis in gastrointestinal tract. All these epithelial cells express junction complex proteins and form cell junctions such as adherens and TJs, although the TJs have small differences among different epithelial cells. The TJs, located most apically on the lateral membrane, are required for the proper formation of epithelial cell polarity as well as sustaining of the mucosal barrier. Furthermore, TJs are the key cell junctions modulating the paracellular pathway. Understanding the diversity of the TJs between intestinal epithelial cells and their different roles in defending pathogens' invasion and modifying the paracellular pathway are attractive to exploration.     

Function and regulation of SPLUNC1 protein in Mycoplasma infection and allergic inflammation

Respiratory infections, including Mycoplasma pneumoniae (Mp), contribute to asthma pathobiology. To date, the mechanisms underlying the increased susceptibility of asthmatics to airway Mp infection remain unclear. Short palate, lung, and nasal epithelium clone 1 (SPLUNC1) protein is a recently described large airway epithelial cell-derived molecule that was predicted to exert host defense activities. However, SPLUNC1 function and regulation in an infectious or allergic milieu are still unknown. We determined host defense and anti-inflammatory functions of SPLUNC1 protein in Mp infection and the regulation of SPLUNC1 by Mp and allergic inflammation (e.g., IL-13). SPLUNC1 function was examined in Mp or human airway epithelial cell cultures by using SPLUNC1 recombinant protein, overexpression and RNA interference. Human and mouse bronchial epithelial SPLUNC1 was examined using immunostaining, Western blotting, ELISA, laser capture microdissection, and real-time PCR. Mouse models of Mp infection and allergic inflammation and air-liquid interface cultures of normal human primary bronchial epithelial cells were used to study SPLUNC1 regulation by Mp and IL-13. We found that: 1) SPLUNC1 protein decreased Mp levels and inhibited epithelial IL-8 production induced by Mp-derived lipoproteins; 2) normal human and mouse large airway epithelial cells expressed high levels of SPLUNC1; and 3) although Mp infection increased SPLUNC1, IL-13 significantly decreased SPLUNC1 expression and Mp clearance. Our results suggest that SPLUNC1 serves as a novel host defense protein against Mp and that an allergic setting markedly reduces SPLUNC1 expression, which may in part contribute to the persistent nature of bacterial infections in allergic airways.     

IL-6-dependent mucosal protection prevents establishment of a microbial niche for attaching/effacing lesion-forming enteric bacterial pathogens

Enteric infections with attaching/effacing lesion-inducing bacterial pathogens are a worldwide health problem. A murine infection model with one such pathogen, Citrobacter rodentium, was used to elucidate the importance of the pleiotropic immune regulator, IL-6, in the pathogenesis of infection. IL-6 was strongly induced in colonic epithelial cells and macrophages upon C. rodentium infection and was required for effective host defense, because mice lacking IL-6 failed to control bacterial numbers 2-3 wk after infection and exhibited increased mortality. IL-6 was not needed for mounting effective T and B cell responses to the pathogens, nor was it important for induction of IFN-gamma or TNF-alpha, cytokines involved in host defense against the bacteria, or the antibacterial effector, NO. Instead, IL-6 played a key role in mucosal protection, since its absence was associated with marked infection-induced apoptosis in the colonic epithelium and subsequent ulcerations. Cell culture studies confirmed that IL-6 protected colon epithelial cells directly against inducible apoptosis, which was accompanied by increased expression of an array of genes encoding antiapoptotic proteins, including Bcl-x(L), Mcl-1, cIAP-2, and Bcl-3. Ulcerations appeared to be pathogenetically important, because bacteria localized preferentially to those regions, and chemically induced colonic ulcerations promoted bacterial colonization. Furthermore, blood components likely present in ulcer exudates, particularly alanine, asparagine, and glycine, promoted bacterial growth. Thus, IL-6 is an important regulator of host defense against C. rodentium by protecting the mucosa against ulcerations which can act as a microbial niche for the bacteria.     

IL-15 up-regulates iNOS expression and NO production by gingival epithelial cells

To investigate the biological activity of epithelial cells in view of host defense, we analyzed the mRNA expression of inducible NOS (iNOS) as well as NO production by human gingival epithelial cells (HGEC) stimulated with IL-15. RT-PCR analysis revealed that HGEC expressed IL-15 receptor alpha-chain mRNA. In addition, stimulation with IL-15 enhanced iNOS expression by HGEC through an increase of both mRNA and protein levels. Moreover, IL-15 up-regulated the production of NO(2)(-)/NO(3)(-), a NO-derived stable end product, from HGEC. The enhanced NO production by IL-15 was inhibited by AMT, an iNOS-specific inhibitor. These results suggest that IL-15 is a potent regulator of iNOS expression by HGEC and involved in innate immunity in the mucosal epithelium.     

Structure and function of intercellular junctions in human cervical and vaginal mucosal epithelia

The mucosal epithelium is a major portal for microbial invasion. Mucosal barrier integrity is maintained by the physical interactions of intercellular junctional molecules on opposing epithelial cells. The epithelial mucosa in the female reproductive tract provides the first line of defense against sexually transmitted pathogenic bacteria and viruses, but little is known concerning the structure and molecular composition of epithelial junctions at this site. In the present study, the distribution of tight, adherens, and desmosomal junctions were imaged in the human endocervix (columnar epithelium) and ectocervix (stratified squamous epithelium) by electron microscopy, and permeability was assessed by tracking the penetration of fluorescent immunoglobulin G (IgG). To further define the molecular structure of the intercellular junctions, select junctional molecules were localized in the endocervical, ectocervical, and vaginal epithelium by fluorescent immunohistology. The columnar epithelial cells of the endocervix were joined by tight junctions that excluded apically applied fluorescent IgG. In contrast, the most apical layers of the ectocervical stratified squamous epithelium did not contain classical cell-cell adhesions and were permeable to IgG. The suprabasal and basal epithelial layers in ectocervical and vaginal tissue contained the most robust adhesions; molecules characteristic of exclusionary junctions were detected three to four cellular layers below the luminal surface and extended to the basement membrane. These data indicate that the uppermost epithelial layers of the ectocervix and vagina constitute a unique microenvironment; their lack of tight junctions and permeability to large-molecular-weight immunological mediators suggest that this region is an important battlefront in host defense against microbial pathogens.